Surface-Induced Dissociation of Homotetramers with D2 Symmetry Yields their Assembly Pathways and Characterizes the Effect of Ligand Binding

Abstract

Understanding of protein complex assembly and the effect of ligand binding on their native topologies is integral to discerning how alterations in their architecture can affect function. Probing the disassembly pathway may offer insight into the mechanisms through which various subunits self-assemble into complexes. Here, a gas-phase dissociation method, surface-induced dissociation (SID) coupled with ion mobility (IM), was utilized to determine whether disassembly pathways are consistent with the assembly of three homotetramers and to probe the effects of ligand binding on conformational flexibility and tetramer stability. The results indicate that the smaller interface in the complex is initially cleaved upon dissociation, conserving the larger interface, and suggest that assembly of a D2 homotetramer from its constituent monomers occurs via a C2 dimer intermediate. In addition, we demonstrate that ligand-mediated changes in tetramer SID dissociation behavior are dependent on where and how the ligand binds.

Bouncing off the Walls: Excited Protein Complexes Tell Their Story

“…This results in nearly symmetrical dissociation, both in terms of charge and topology. Quintyn et al (2015) show that SID dissociation is determined by the relative stability (and thus also the size) of interaction surfaces -thereby resembling the hierarchy and pathways of subunit assembly.…”

“…As a consequence, virtually no unfolding or charge redistribution are observed, resulting in dissociation products that are reflective of the topology of the complex studied (Jones et al, 2006). In the work by Quintyn et al (2015), the authors study the dissociation pathways of three tetrameric protein complexes with D2 symmetry, with and without additional ligands bound. They show that SID, unlike CID, proceeds via cleavage of the smallest dimer-dimer interface.…”

“…Advances such as the surface-induced dissociation (SID) method described by Quintyn et al (2015), however, are not as contradictory as they first sound, given that the controlled disassembly of macromolecular complexes gives us an opportunity to understand how the specific parts work together. The ability to gently release proteins from the native-like environment of a buffered, aqueous solution is owed to the use of nano-electrospray ionization, in combination with careful tuning of the interface and mass analyzer of the instrument (in particular acceleration voltages and gas pressures).…”

“…In this issue of Chemistry & Biology, Quintyn et al (2015) show that modified instruments capable of surface-induced dissociation allow dissection of protein complexes in a way that is reminiscent of their native topology and architecture.…”

Ion Mobility Spectrometry-Hydrogen Deuterium Exchange Mass Spectrometry of Anions: Part 3. Estimating Surface Area Exposure by Deuterium Uptake

“…Although the concept of comparisons of CCS values for biomolecular ions was established nearly a decade earlier [28–31], theoretical approaches for the generation of trial structures for extremely large ions have proceeded at a rapid pace [32]. One notable advantage of the IMS-MS approach for protein complex studies is that collisional activation can be used to study protein complex, subcomplex, and subunit ions [23, 33, 34]. A limitation of IMS-MS studies is that only general information regarding ion shape is obtained.…”